Napovedovanje učinkovitosti in varnosti zdravljenja z azatioprinom pri pacientih s Crohnovo boleznijo

Kuhar, Mitja (author), Mlinarič-Raščan, Irena (mentor), Drobne, David (comentor)

Crohnova bolezen je ena od najpogostejših oblik avtoimunske kronične vnetne črevesne bolezni. Pomemben terapevtski pristop za vzdrževanje bolezni v fazi remisije je zdravljenje z azatioprinom. Zaradi ozkega terapevtskega okna te imunosupresivne zdravilne učinkovine se lahko med terapijo razvijejo hujši neželeni učinki, kot sta mielosupresija in hepatotoksičnost. V izogib temu je pomembno načrtovanje učinkovite in varne terapije z določitvijo ustreznega individualnega odmerka azatioprina za bolnika na osnovi sledečih diagnostičnih pristopov: genotipizacija ter merjenje aktivnosti encima tiopurin S-metiltransferaze (TPMT) pred zdravljenjem ter določitev koncentracije glavnih presnovkov azatioprina – 6-tiogvanozinskih nukleotidov (6-TG) in 6-metilmerkaptopuina (6-MMP) – 4–6 tednov po začetku terapije. Magistrsko delo je zajemalo analize na vzorcih krvi bolnikov s Crohnovo boleznijo, ki so bili napoteni iz UKC Ljubljana, oddelka za gastroenterologijo, v Laboratorij za molekularno diagnostiko na Fakulteti za farmacijo z namenom določitve genotipa in aktivnosti TPMT ter analize glavnih presnovkov zdravila. Po izolaciji DNA iz krvi bolnikov smo izvedli genotipizacijo najpogostejših alelov TPMT (TPMT *2, *3A, *3C), ki je temeljila na reakciji PCR z uporabo hidrolizirajočih sond. Na delu vzorcev smo v lizatih rdečih krvničk na osnovi pretvorbe 6-merkaptopurina v 6-MMP s pomočjo postavljene metode HPLC nadalje določili aktivnost encima TPMT. Identificirali smo prisotnost najpogostejših alelnih variant TPMT*3A in TPMT*3C, in sicer pri 6,25 % oz. 1,56 % preiskovancev. Na predhodno določenih presnovkih azatioprina (6-TG in 6-MMP) smo preučili tudi vpliv genotipa TPMT na glavna presnovka ter na njuno razmerje. Prisotnost variantnega alela TPMT je bila značilno povezana z višjimi koncentracijami 6-TG ter z nizkim razmerjem 6-MMP : 6-TG. Nadalje smo preverili napovedno vrednost za pojav neželenih učinkov, ki je temeljila na koncentracijah 6-TG, 6-MMP ter njunega razmerja. Po literaturnih podatkih koncentracija 6-TG > 450 pmol x (8 x 10^8 eritrocitov)^-1 korelira s povečanim tveganjem za mielosupresijo, koncentracija 6-MMP > 5700 pmol x (8 x 10^8 eritrocitov)^-1 ter razmerje 6-MMP : 6-TG > 11 pa s hepatotoksičnostjo. Na naši populaciji je imel vsak tretji preiskovanec prisoten vsaj en dejavnik tveganja za pojav neželenih učinkov azatioprina pri prvem odvzemu vzorca. Mielosupresija bi se lahko izrazila pri 20,31 % preiskovancev, hepatotoksičnosti pa bi bilo podvrženih 23,44 % preiskovancev. Z merjenjem metabolitov tekom terapije je te vrednosti pri večini bolnikov ob uvedbi ustreznih ukrepov v terapijo zdravnikom uspelo znižati. Pomen pravega odmerjanja in spremembe zdravljenja na osnovi genotipizacije TPMT oz. merjenja metabolitov smo ponazorili na dveh kliničnih primerih. V primeru mielotoksičnosti se je odmerjanje azatioprina zamenjalo z biološkim zdravilom, pri hepatotoksičnosti pa se je po smernicah ob uvedbi alopurinola znižal odmerek azatioprina. Rezultati tega magistrskega dela poudarjajo pomen določanja genotipa in aktivnosti TPMT in podrobno spremljanje metabolitov tiopurinov tekom zdravljenja. S temi diagnostičnimi pristopi lahko preprečimo resne neželene učinke ali sprejmemo ustrezne ukrepe, ki vodijo v remisijo bolezni brez resnejših zapletov.

• Language: Slovenian
• Keywords: Crohnova bolezen, azatioprin, tiopurin S-metiltransferaza, 6-tiogvanin, 6-metilmerkaptopurin
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-129804
• Publication date in RUL: 08.09.2021

KUHAR, Mitja, 2021, Napovedovanje učinkovitosti in varnosti zdravljenja z azatioprinom pri pacientih s Crohnovo boleznijo [online]. Master’s thesis. [Accessed 20 July 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=129804

Secondary language

• Language: English
• Title: Efficacy and safety prediction of azathioprine therapy in patients with Crohn's disease

Abstract

Crohn's disease is one of the most common forms of autoimmune chronic inflammatory bowel diseases. An important therapeutic approach to maintain the disease in remission is azathioprine therapy. Due to the narrow therapeutic window of this immunosuppressive drug, severe side effects, such as myelosuppression and hepatotoxicity, may develop during therapy. To avoid this, it is important to plan an effective and safe therapy by determining the appropriate individual dose of azathioprine for the patient based on the following diagnostic approaches: genotyping and measurement of the activity of the enzyme thiopurine S-methyltransferase (TPMT) prior to treatment, and determination of the levels of the main metabolites of azathioprine - 6-thioguanosine nucleotides (6-TG) and 6-methylmercaptopurine (6-MMP) – 4–6 weeks after the initiation of therapy. The master's thesis included analyses on blood samples from Crohn's disease patients referred from the UKC Ljubljana, Department of Gastroenterology to the Laboratory of Molecular Diagnostics at the Faculty of Pharmacy in order to determine the genotype and activity of TPMT and to analyse the major metabolites of the drug. After DNA isolation from the patients' blood, genotyping of the most frequent TPMT alleles (TPMT *2, *3A, *3C) was performed based on PCR using hydrolysable probes. In a subset of samples, TPMT enzyme activity was further determined in red blood cell lysates based on the conversion of 6-mercaptopurine to 6-MMP using an established HPLC method. The presence of the most common allelic variants TPMT*3A and TPMT*3C was identified in 6.25% and 1.56% of the subjects, respectively; the most common allelic variants TPMT*3A and TPMT*3C were identified in 6.25 % and 1.56 % of the subjects. We also investigated the influence of the TPMT genotype on the major metabolites and their ratio on previously determined azathioprine metabolites (6-TG and 6-MMP). The presence of a variant TPMT allele was typically associated with higher 6-TG concentrations and a low 6-MMP : 6-TG ratio. We further tested the predictive value for the occurrence of adverse effects based on 6-TG, 6-MMP concentrations and their ratio. According to the literature, a 6-TG concentration > 450 pmol x (8 x 10^8 erythrocytes)^-1 correlates with an increased risk of myelosuppression, and a 6-MMP concentration > 5700 pmol x (8 x 10^8 erythrocytes)^-1 and a 6-MMP : 6-TG ratio > 11 with hepatotoxicity. In our population, one in three subjects had at least one risk factor for azathioprine adverse effects present at the first sample collection. Myelosuppression could be manifested in 20.31 % of subjects and hepatotoxicity in 23.44 % of subjects. By measuring metabolites during the course of therapy, physicians were able to lower these values in most patients when appropriate measures were introduced into the therapy. The importance of correct dosing and treatment modification based on TPMT genotyping or metabolite measurement was illustrated in two clinical cases. In the case of myelotoxicity, the dosage of azathioprine was switched to a biologic, and in the case of hepatotoxicity, the dose of azathioprine was reduced according to guidelines when allopurinol was introduced. The results of this Master thesis underline the importance of genotyping and activity determination of TPMT and close monitoring of thiopurine metabolites during treatment. These diagnostic approaches can prevent serious side effects or take appropriate measures leading to disease remission without serious complications.

• Keywords: Crohn's disease, azathioprine, thiopurine S-methyltransferase, 6-thioguanine, 6-methylmercaptopurine